1. Field of Invention
The invention describes methods to prepare clear solutions of diterpenes, such as Forskolin and its congeners that are sparingly soluble or insoluble in water, of concentrations 0.09% to 6%, for convenient use in ophthalmic preparations as well as in topical, oral, injectable and other dosage forms, for human and veterinary use.
2. Description of Prior Art
Certain active pharmaceutical ingredients are inherently insoluble or very sparingly soluble in water or in aqueous vehicles. Very often their intended use may require their application in water or in aqueous vehicles. To achieve therapeutically active concentrations of such water insoluble active pharmaceutical ingredients in stable form has always been actively pursued. While the technique of molecular structural manipulation of the active pharmaceutical ingredient that is insoluble in water could be adopted, incorporating structural features that promote aqueous solubility may result in the attenuation or modification of the intended desired pharmacological properties. Hence it maybe most desirable to invent methods of solubilizing the active ingredients in their native structural form by other means.
Aqueous solubility of drugs is a desirable feature from many angles. Aqueous formulations are sterilizable by standard techniques such as filtration etc to render such preparations suitable for systemic administration. Also aqueous preparations are preferable in dermatological, gynecological, otological, rhinological and on mucous membrane applications. Especially useful are aqueous ophthalmic preparations of drugs.
Forskolin (CAS no 66575-29-9) is a naturally occurring labdane diterpene from Coleus forskohlii (Bhat, S. V.; Bajwa, B. S.; Dornauer, H.; de Souza, N. J.; Fehlabar, H.-W.; Tetrahedron Lett., (1977), 18, 1669). It has several desirable pharmacological properties.
Forskolin displays positive inotropic, antihypertensive and broncho-spasmolytic activity; (Bhat, S. V.; Dohadwalla, A. N.; Bajwa, B. S.; Dadkar, N.; Dornauer, H.; de Souza, N. J.; J Med Chem., (1983), 26, 486).
It lowers intraocular pressure (Caprioli J, Sears M.; Lancet (1983); April 30;1(8331):958-60;. Badian M et al.; Klin Monatsbl Augenheilkd (1984);185:5226, Zeng S, et al. Yan Ke Xue Bao (1995);11:173-176, Lee P Y, et al.; Arch Ophthalmol (1987);105:249-252,. Meyer B H, et al. S Afr Med J. (1987);71:570-571; Seto C, et al.; Jpn J Ophthalmol (1986);30:238-244.; Burstein N I et al. Exp Eye Res (1984);39:745-749; Brubaker R F et al. Arch Ophthalmol (1987); 105:637-641).
Diverse biological activities are observed by raising the levels of cAMP, and as a result activating protein kinase. Such properties have led to numerous uses of Forskolin. Due to such activities, more than 1500 citations dealing with the physiological properties of Forskolin appeared in Chemical Abstracts in 2001. However, Forskolin is highly insoluble in water.
Intensive efforts have been made on the molecular manipulation of Forskolin to make such derivatives of Forskolin as will be water soluble. Such attempts have always met with mixed success (Lal, B.; Gangopadhyay, A. K.; Rajagopalan, R.; Ghate, A. V.; Bioorganic & Medicinal Chemistry, (1998), 6(11), 2061-2073; Lal, B.; Gangopadhyay, A. K.; Gidwani, R. M.; Fernandez, M.; Rajagopalan, R.; Ghate, A. V.; Bioorganic & Medicinal Chemistry, (1998), 6(11), 2075-2083).
As an alternative to chemical manipulations of the drug molecular structure, physicochemical techniques of enhancing the solubility of the underivatized drug in water have been employed. Notable technologies include micellar solubilization using surface active ingredients, which will form water soluble micelles containing the drug. Another related technique is complexation of the drug molecule with a host molecule. The host molecule is usually one that has good solubility in water. The host molecule does not form any covalent bonds with the drug molecule but forms a weak complex through non-covalent interactions and the host molecule(s) keep the drug molecule(s) in water solution.
Cyclodextrins are cyclic oligosaccharides which have been recognized as useful pharmaceutical excipients. The common cylcodextrins are called α-, β-, γ- and δ-cyclodextrins depending on the number of glucose molecules in the cyclic oligosaccharide structure. These cyclodextrins are (α1, 4)-linked oligosaccharides of α-D-glucopyranose containing a relatively hydrophobic central cavity and hydrophilic outer surface. These molecules are not exactly perfect cylinders due to restriction of completely free rotation about their linking bonds of the units of the sugar molecule. They assume the shape of a torus or a truncated cone. The secondary hydroxyl groups line the wider edge of the rim while the primary hydroxyl groups line the narrow side of the torus. The solubilities of these molecules in water and the diameter of the central cavity have been known and published (Loftsson, T.; Brewster, M. E.; J Pharmaceutical Sciences, (1996), 85, 1017 & Rajewski, R. A.; Stella, V. J.; J Pharmaceutical Sciences, (1996), 85, 1142). The structure of β-cyclodextrin containing seven glucose units is shown as an example 
The α-cyclodextrin has six anhydroglucose molecules in the ring; the γ- and δ-cyclodextrins have eight and nine respectively. The α-, β-, γ- and δ-cyclodextrins have their water solubilties at 25° C. (g/100 ml) 14.5, 1.85, 23.2 & 8.19 respectively. The α-, β-, γ- and δ-cyclodextrins are sometimes called natural cyclodextrins and their solubilities in water are at the lower end of the desirable range. Nevertheless they proved very good solubilizing agents for some of the water insoluble molecules. To increase the aqueous solubilities of these natural cyclodextrins, molecular modifications of these α-, β-, γ- and δ-cyclodextrins have been carried out in the literature.
These modified cyclodextrins have much higher solubilities than their natural counterparts and they can be classified as Methylated derivatives of β-cyclodextrin, 2-hydroxypropylated β- and γ-cyclodextrins, sulfobutylated-β-cyclodextrins, branched cyclodextrins, acylated β- and γ-cyclodextrins.
The cyclodextrins can be methylated by Kuhn-Trischmann methylation, Wacker”s industrial method with methyl chloride under pressure and Hakamori methylation using methylhalogenide and sodium hydride (see, Szente, L.; Szejtli, J.; Advanced Drug Delivery Reviews, (1996), 36, 17). The first two technologies have been used to produce randomly methylated cyclodextrin mixture. On the other hand Hakamori methylation is reported to produce a fully methylated heptakis 2,3,6-tri-O-methylated cyclodextrins. The introduction of methyl substituents in the place of the hydrogens of the hydroxy group of parent β-cyclodextrin dramatically improves the solubility of this randomly methylated cyclodextrin, referred in this invention as RAMEBCD versus the parent β-cyclodextrin.
There are totally 21 hydroxyl groups (14 secondary hydroxyl groups and seven primary hydroxyl groups) in β-cyclodextrin. The aqueous solubility of RAMEBCD increases as the number of methyl groups reaches around 13-14 and decreases as methylation approaches 21 methoxy groups per molecule of β-cyclodextrin. An example of a commercially available RAMEBCD product can be cited the one produced by Wacker Chemie and marketed under the name CAVASOL® W7 M Pharma (CAS no 128446-36-6). Aqueous solubilities of such RAMEBCDs are typically ˜220 g/100 ml of water. Such RAMEBCDs have an average degree of methylation ˜1.7 to 1.9 per anhydroglucose unit. Such RAMEBCDs are available commercially and have very good aqueous solubilities as noted. The general structure of such RAMEBCDs are shown as follows 
Reacting cyclodextrins with propylene oxide in alkaline solution results in substitution of the hydroxy groups in the cyclodextrins with 2-hydroxypropyl derivatives. A higher substitution of the hydroxyls with propylene oxide also results in the formation of oligomeric hydroxypropylene oxide side chain formation. Such 2-hydroxy-propyl-β-cyclodextrin referred in this invention as HPBCD is represented by the following generic structure. Such materials are available commercially. 
Similarly to HPBCD, γ-cyclodextrin can be hydroxypropylated to give hydroxypropyl γ-cyclodextrin, referred as HPGCD in this invention. Such materials are available commercially 
A review on the applications of cyclodextrin in the ophthalmic field has appeared (Loftssona, T.; Jarvinen, T.; Advanced Drug Delivery Reviews, (1999), 36, 59). A patent, U.S. Pat. No. 6,346,273 describes the aqueous solubilization of forskolin through the use of polyvinylpyrrolidone and a surfactant, polyethyleneglycol-glyceryl tririicinoleate. The maximum solubility of Forskolin achieved in this patent is 0.2%.
U.S. Pat. No. 4,476,140 describes a composition and method for treatment of Glaucoma by administration of a therapeutically effective amount of a material selected from the group consisting of forskolin, colforsin and polyoxygenated Labdane derivatives. The active agent concentration of 0.1% to 4% is reported herein to be physiologically effective when administered as a topical suspension to the eye.
U.S. Pat. Nos. 5,070,209, 4,978,678, 5,023,344, 4,871,764 describe novel 12-halogenated forskolin derivatives, intermediates and processes for the preparation thereof, and methods for reducing intraocular pressure utilizing compounds or compositions.
EP0268256 describes novel 12-halogenated forskolin derivatives, intermediates and processes for their preparation, and methods for reducing intraocular pressure utilizing the compounds or compositions.
However these prior art references do not describe solubilization of unmodified forskolin to obtain clear aqueous solutions of concentrations of 1% or greater.